Telomerase activity is linked with cellular immortality and tumorigenesis. It is responsible for the elongation of chromosomal DNA through addition of repetitive (TTAGGG)n guanine-rich sequences to the 3'-single- stranded telomeric overhang. The guanine-rich sequences can fold in vitro to form G-quadruplexes structures, and can inhibit telomerase providing a potential target for controlling tumorigenesis. However, development of quadruplex interactive agents (QIAs) that stabilize the quadruplex structure for clinical application remains slow, because of the polymorphic conformation of quadruplexes in solution and potential quadruplex multimerization along the telomeric DNA. A significant increase in knowledge of the fundamental chemical and physical rules that govern DNA quadruplex conformation and stabilization is thus required for rational development of QIAs for chemotherapeutic applications. The overall goal of the proposed research focuses on conformation and multimerization of G-quadruplexed DNA under physiological conditions. The objectives of the application focus on: 1) examining the effects of varying solution conditions on quadruplex conformational stability and its modulation on binding model QIA ligands;and 2) to examine conformational driving forces involved in multimeric quadruplex formation and the effects of QIA interactions. We have developed a novel family of fluorescently labeled human telomeric sequences (h(TTAGGG)4) through site-specific replacement of single guanine residues using a fluorescent guanine analog (6-MI). In combination with CD-measurements, electrophoretic and UV approaches for characterization studies, these fluorescent telomeric sequences can provide spectroscopic information about local environmental effects at unique guanine sites along the human quadruplex sequence and when in the folded conformation. The information provided will allow us to begin to understand the role of conformational heterogeneity of individual guanine residues in stabilizing and modulating the quadruplex conformation and multimeric formation in solution, and to use this information for rational design of QIAs with high quadruplex binding selectivity and affinity. PUBLIC HEALTH RELEVANCE: The focus of this proposal is on sequences of DNA that fold into a four-stranded structure called a quadruplex and can inhibit the activity of a key protein called telomerase, which is linked with cancer. The proposed studies will provide information on what influences shape and organization of the quadruplex particularly in solution. This basic knowledge is critically important as it will allow us to better design Quadruplex Interactive Agent (QIA) therapeutics that bind more effectively and selectively and promote quadruplex folding of the DNA in order to inhibit telomerase and control tumorigenesis.